Thiopurines are in widespread clinical use for the treatment of immunological disorders and certain cancers. However, treatment failure due to resistance or adverse drug reactions are common, asking for new therapeutic strategies. We investigated the potential of 6-thioguanosine monophosphate (6sGMP) prodrugs to overcome resistance to 6-thioguanine. We successfully developed synthetic routes toward diverse 6sGMP prodrugs, tested their proliferation inhibitory potential in different cell lines, and examined their mode of action. Our results show that 4-acetyloxybenzyl- and cycloSaligenyl-derivatized 6sGMP prodrugs are effective antiproliferative compounds in cells that are resistant to thiopurines. We find that resistance is related to the expression of salvage pathway enzyme HGPRT. Using TUC-seq DUAL, we demonstrate the intracellular conversion of 6sGMP prodrugs into bioactive 6sGTPs. Thus, our study offers a promising strategy for thiopurine therapy by using 6sGMP prodrugs, and it suggests TUC-seq DUAL as a simple and fast method to measure the success of thiopurine therapy.